Detection of serum proteins by native polyacrylamide
gel electrophoresis using Blue Sepharose CL-6B-containing
Haruhiro Muratsubaki,a,*Kaoru Satake,bYasuhisa Yamamoto,cand Keiichiro Enomotoa
aDepartment of Clinical Biochemistry, Kyorin University School of Health Sciences, Miyashita, Hachioji, Tokyo 192-0005, Japan
bKeihin Health Center, Nippon Kohkan Hospital, Watarida, Kawasaki, Kanagawa 210-0855, Japan
cCriminal Investigation Laboratory, Saitama Prefectural Police Headquarters, Uetake, Ohmiya, Saitama 330-0036, Japan
Received 28 February 2002
Analysis of serum proteins by native polyacrylamide gel electrophoresis is difficult because albumin is abundant in serum and
interferes with the resolution of other proteins, especially a-antitrypsin which has a mobility that is very similar to that of albumin.
We present here a method in which serum proteins are separated by polyacrylamide gel electrophoresis using stacking gels con-
taining Blue Sepharose CL-6B, which has a high affinity for albumin, lipoproteins, kinases, and pyridine-nucleotide-dependent
oxidoreductases. During electrophoresis, proteins that bind to Blue Sepharose CL-6B stay in the stacking gel and do not migrate
into the separating gel. As a consequence, certain proteins, including a1-antitrypsin, can be detected as clear bands. This method
overcomes the requirement for fractionation of serum samples prior to electrophoresis to remove albumin and allows the simul-
taneous analysis of many samples. ? 2002 Elsevier Science (USA). All rights reserved.
Serum is a very complex mixture of proteins. Initial
biological functions of individual proteins from serum is
frequently accomplished by the use of native polyacryl-
amide gel electrophoresis (PAGE). However, using this
method, it can be difficult to isolate pure protein without
contamination with albumin, the serum protein that is
present in the highest mass concentration. Furthermore,
albumin can interfere with the qualitative detection of
other serum proteins such as a1-antitrypsin. Thus, albu-
min-free serum is often used for PAGE and is usually
obtained by passing serum samples through Blue Sepha-
rose CL-6B columns [1–3]. This relies on the affinity of
albumin, along with lipoproteins, kinases, and pyridine-
nucleotide-dependent oxidoreductases, for Cibacron
Blue F3G-A, the ligand of Blue Sepharose CL-6B [4–8].
In the present study, we describe a simple method for
detecting serum proteins by native PAGE in which Blue
Sepharose CL-6B is incorporated into the stacking gel to
remove proteins with affinity for Cibacron Blue F3G-A.
Materials and methods
PAGE in the absence of denaturing reagent was
performed using an Atto Rapidas Slab Gel Electro-
phoresis system (Atto Corp.) according to the manu-
facturer’s instructions. Slab gels consisted of a 7.5%
acrylamide resolving gel (13:5 ? 10 ? 0:2cm) and a 4.5%
stacking gel (13:5 ? 3:5 ? 0:2cm) . The PAGE meth-
od using a stacking gel containing Blue Sepharose CL-
6B is illustrated in Fig. 1. The stacking gel containing
Blue Sepharose CL-6B was constructed on top of the
resolving gel as follows. Blue Sepharose CL-6B (0.4g)
was gently mixed in 4.5ml of stacking gel mixture by
swirling. Without delay, the slab gel plate was filled with
the gel mixture to a height of 1.5cm. To avoid sedi-
mentation of the Sepharose, the volume of ammonium
persulfate was adjusted to obtain gel polymerization
within 2min. After polymerization, the stacking gel
mixture without Blue Sepharose CL-6B was poured on
top and a comb was inserted immediately to construct
sample wells. Samples were prepared by mixing 10ll of
serum with 40ll of 10mM Tris–HCl buffer (pH 6.8)
containing 20% glycerol. Twenty microliters of this
Analytical Biochemistry 307 (2002) 337–340
*Corresponding author. Fax: +81-426-91-1094.
E-mail address: firstname.lastname@example.org (H. Muratsubaki).
0003-2697/02/$ - see front matter ? 2002 Elsevier Science (USA). All rights reserved.
sample was loaded into a well and initially electropho-
resed at a current of 20mA. After the bromphenol blue
dye front had moved about 1cm into the resolving gel,
the current was increased to 40mA. When the dye front
reached the bottom of the gel, electrophoresis was
stopped. The gel was then stained with Coomassie
brilliant blue R250 for protein detection. To quantify
levels of a1-antitrypsin, the gel was scanned with a
densitometer (Johoko, Tokyo) containing a yellow filter
To identify serum proteins, immunoblotting was
performed using an Atto Horizon Blot system (Atto
Corp.) according to the manufacturer’s instructions .
Following electrophoresis, proteins were electroblotted
onto a nitrocellulose membrane. The membrane was
then incubated with the primary antibodies and then
treated with the respective secondary antibodies conju-
gated with alkaline phosphatase. Bands were visualized
using nitroblue tetrazolium and 5-bromo-4-chloro-3-
Blue Sepharose CL-6B was purchased from Phar-
macia; anti-human haptoglobin and anti-human trans-
ferrin were from ICN Immuno Biologicals; albumin and
a1-antitrypsin were from Sigma; anti-human a1-anti-
trypsin and anti-human ceruloplasmin were from Wako
Pure Chemical Industries; and goat anti-rabbit IgG
conjugated with alkaline phosphatase was from Bio-
Rad Laboratories. All other chemicals were of reagent
Results and discussion
Normal human serum was subjected to native PAGE
using a stacking gel without Blue Sepharose CL-6B. As
shown in Fig. 2, a1-antitrypsin and albumin were in-
completely resolved. On the other hand, when electro-
phoresis was carried out using a stacking gel containing
Blue Sepharose CL-6B, a1-antitrypsin appeared as a
single clear band. Thus, the method improved resolution
because interfering proteins, including albumin, which
have an affinity for Cibacron Blue F3G-A, are adsorbed
by the stacking gel and do not migrate into the resolving
gel. Furthermore, the electrophoretogram generally
showed sharp, clear bands because of the decreased
The identification of protein bands after electropho-
resis was performed by comparing them with the elec-
trophoretic pattern of each pure protein and by
immunoblotting. Using our method, we were able to
easily identify a1-antitrypsin, ceruloplasmin, transferrin,
and haptoglobin (Fig. 2).
During the metabolic response to injury and infec-
tion, the serum concentrations of the acute-phase pro-
haptoglobin are markedly increased . We studied the
electrophoretograms of sera from patients with inflam-
matory diseases and sera obtained from patients fol-
lowing surgical operations and labor. The sera from
these patients showed the characteristic inflammatory
response of increased levels of acute-phase proteins,
which is used for diagnosis and clinical management
(Fig. 3). In response to acute inflammation, a1-anti-
trypsin was increased in most of the patients examined.
In one patient with infectious disease, ceruloplasmin,
which is synthesized in hepatocytes, was markedly
Fig. 1. Scheme of preparation of native PAGE gels with Blue Sepha-
rose CL-6B present in the stacking gel.
Fig. 2. Separation of serum proteins by native PAGE with (A) or
without (B) Blue Sepharose CL-6B present in the stacking gel. Lane 1,
4ll of serum. Lane 2, 5lg of a1-antitrypsin. Lane 3, 20lg of albumin.
Lane 4, 5 lg of a1-antitrypsin and 20lg of albumin. Lane 5, 4ll of
serum and 5lg of a1-antitrypsin. HG, haptoglobin; TF, transferrin;
CP, ceruloplasmin; AT, a1-antitrypsin.
H. Muratsubaki et al. / Analytical Biochemistry 307 (2002) 337–340
increased (see lane 7). We detected a protein showing a
electrophoretic mobility that was slightly faster than
that of a1-antitrypsin in sera from patients with liver
cancer (lanes 2 and 10). These patients suffer from
hyperbilirubinemia. The binding of human serum al-
bumin to Blue Sepharose is reported to diminish with
increasing concentration of bilirubin . The addition
of unconjugated bilirubin to normal serum or purified
albumin resulted in the appearance of the protein in the
same region of the gel as the protein from the serum of
liver cancer patients. Thus, this protein was concluded
to be bilirubin-bound albumin. The a1-antitrypsin band
in lane 9 appears to have a slightly faster migration time
than a1-antitrypsin in other lanes. a1-Antitrypsin shows
considerable genetic variability . Accordingly, the
anomalous migration of the band in lane 9 may be due
to a genetic variant with a low rate of incidence in the
Japanese population. Also, three haptoglobin serotypes
are found in serum . The haptoglobin identified here
is one of these three types (Fig. 2A; lanes 3, 5, 9, 11, 14,
and 16 in Fig. 3). However, the other types could not be
identified, because of poor resolution of proteins that
move more slowly than the type identified, as described
We examined quantitatively the level of a1-antitryp-
sin by the method presented here. Various volumes of
serum were electrophoresed and the maximum absor-
bance of the a1-antitrypsin band at 610nm was meas-
ured usinga densitometer.
established for serum volumes between 0.5 and 10ll, a
range suitable for clinical study with the equipment and
detection system used here (data not shown). When 4ll
of albumin solution (150g/L) was loaded into the well
(6mm in width and 2mm in thickness), most of the
added albumin was removed by the stacking gel con-
taining Blue Sepharose. This is far higher than the
concentration observed in clinical samples (<60g/L).
We determined the concentration of a1-antitrypsin in
serum from three healthy subjects and nine patients with
inflammatory disease and compared these values to
those obtained using nephelometry [15,16] (Fig. 4). The
correlation coefficient was 0.935,
It should be emphasized that the preparation of the
stacking gel containing Blue Sepharose must be carefully
carried out. Acrylamide polymerization must be finished
before the Sepharose has time to settle out. The rate
of polymerization was controlled by adjusting the
Fig. 3. Electrophoretogram of normal sera and sera from various patients. Lanes 3 and 9, normal sera. Lanes 2 and 10, sera from patients with liver
cancer. Lanes 7, 11, and 16, sera from patients with infectious diseases. Lanes 1, 4–6, 8, 12–15, 17, and 18, sera from patients following surgical
operations and labor. HG, haptoglobin; TF, transferrin; CP, ceruloplasmin; AT, a1-antitrypsin.
H. Muratsubaki et al. / Analytical Biochemistry 307 (2002) 337–340
concentration of ammonium persulfate, which was used
at nine-fold higher concentration than that commonly
employed. Ammonium persulfate was added without
prior degassing of the gel solution, and the stacking gel
solution was rapidly poured with continual swirling
onto the top of the resolving gel. Swirling the gel mix-
ture prior to pouring is essential in order to avoid
polymerization and for successful preparation of the
stacking gel. The resolution and gel-to-gel reproduc-
ibility of proteins that move more slowly than hapto-
globin on the gels are poor. Further studies will be
required to optimize the stacking gel.
The quantity of available protein for examination
using PAGE is occasionally limited, making the analysis
of low-abundance proteins difficult. Sensitivity can be
enhanced, by passing the serum through a column of
Blue Sepharose CL-6B before electrophoresis, to remove
albumin and enrich the remaining proteins [1–3]. How-
ever, this method requires a large volume of serum and
is cumbersome when large numbers of samples need
processing. On the other hand, using the method de-
veloped by us, many samples can be electrophoresed
simultaneously on the same gel under identical condi-
tions, thereby increasing the reproducibility and com-
parability of the separation patterns. Moreover, with
this method, a large volume of serum can be loaded onto
the gel because albumin is removed, leading to easier
detection of low-abundance proteins in serum. Our
method should also be applicable for other biological
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Fig. 4. Comparison of the results obtained by nephelometry and native
PAGE with Blue Sepharose CL-6B present in the stacking gel for
determination of the concentration of a1-antitrypsin.
H. Muratsubaki et al. / Analytical Biochemistry 307 (2002) 337–340